Niche breadth and divergence in sympatric cryptic coral species (Pocillopora spp.) across habitats within reefs and among algal symbionts

Abstract While the presence of morphologically cryptic species is increasingly recognized, we still lack a useful understanding of what causes and maintains co‐occurring cryptic species and its consequences for the ecology, evolution, and conservation of communities. We sampled 724 Pocillopora corals from five habitat zones (the fringing reef, back reef, and fore reef at 5, 10, and 20 m) at four sites around the island of Moorea, French Polynesia. Using validated genetic markers, we identified six sympatric species of Pocillopora, most of which cannot be reliably identified based on morphology: P. meandrina (42.9%), P. tuahiniensis (25.1%), P. verrucosa (12.2%), P. acuta (10.4%), P. grandis (7.73%), and P. cf. effusa (2.76%). For 423 colonies (58% of the genetically identified hosts), we also used psbA ncr or ITS2 markers to identify symbiont species (Symbiodiniaceae). The relative abundance of Pocillopora species differed across habitats within the reef. Sister taxa P. verrucosa and P. tuahiniensis had similar niche breadths and hosted the same specialist symbiont species (mostly Cladocopium pacificum) but the former was more common in the back reef and the latter more common deeper on the fore reef. In contrast, sister taxa P. meandrina and P. grandis had the highest niche breadths and overlaps and tended to host the same specialist symbiont species (mostly C. latusorum). Pocillopora acuta had the narrowest niche breadth and hosted the generalist, and more thermally tolerant, Durusdinium gynnii. Overall, there was a positive correlation between reef habitat niche breadth and symbiont niche breadth—Pocillopora species with a broader habitat niche also had a broader symbiont niche. Our results show how fine‐scale variation within reefs plays an important role in the generation and coexistence of cryptic species. The results also have important implications for how niche differences affect community resilience, and for the success of coral restoration practices, in ways not previously appreciated.


| INTRODUC TI ON
The ability of biological communities to adjust to changing environmental conditions, and the success of conservation and management interventions, depends on there being a portfolio of species and genomes that promote resilience and adaptive potential (Baums et al., 2022;Colton et al., 2022;Matz et al., 2020;McManus et al., 2021;Webster et al., 2017).A critical and nontrivial aspect of this endeavor is the organization of genetic diversity into species (Bickford et al., 2007;Pante et al., 2015;Ramírez-Portilla et al., 2022;Sheets et al., 2018).Genomic techniques over the past few decades have drastically reshaped our understanding of species boundaries and their permeability to gene flow in natural populations (Bridge et al., 2023;Cowman et al., 2020;Fišer et al., 2018;Stanton et al., 2019).Especially in the case of corals, cryptic species are being rapidly discovered using genomic techniques that have identified evolutionarily distinct lineages, as well as hybrid or introgressed lineages that are not always congruent with current species descriptions relying on morphological differences (e.g., Bongaerts et al., 2021;Bridge et al., 2023;Oury et al., 2023;Pinzón et al., 2013;Schmidt-Roach et al., 2014;Voolstra et al., 2023).Incorrectly grouping morphologically similar individuals from genetically divergent lineages will provide misleading estimates of biodiversity, population connectivity, and adaptive potential, which in turn makes it challenging to make scientifically informed decisions about coral conservation and restoration.While the presence of morphologically cryptic species is increasingly recognized, we still lack a useful understanding of what causes and maintains co-occurring cryptic coral species within reefs.Specifically, we need to know the diversity of resources used and environments tolerated (niche breadth) and the extent to which these differ among species (niche overlap), as well as differences in the identity of symbionts that could underlie holobiont adaptation.
Much of the coral's nutrition relies on the photosynthetic algae to convert inorganic carbon and nitrogen to nutrients through photosynthesis and absorption of nitrogen wastes.The environmental sensitivity of this feedback may differ among algal species and genotypes, or among specific combinations of coral host and algal species or genotypes to influence holobiont fitness (Cornwell et al., 2021;Hoadley et al., 2021;Kemp et al., 2023;Starko et al., 2023;Wall et al., 2020).Because symbiotic Symbiodiniaceae live inside coral cells, they are identified using genetic markers (LaJeunesse et al., 2018).Historically there has been a lack of consensus regarding the organization of genetic data into species, which has slowed progress on understanding how the environment shapes the distribution of symbiont and host genetic diversity, but recent advances in Symbiodiniaceae genomics have led to a growing consensus on how to interpret the genetic diversity in this group (Davies et al., 2023).A particularly valuable system to understand the environmental sensitivity of morphologically similar yet genetically divergent species is corals in the genus Pocillopora.Pocilloporid corals are distributed across contiguous habitat zones within reefs in the Indo-Pacific, and often dominate reefs in terms of percent cover (Edmunds et al., 2016;Pérez-Rosales et al., 2021).Our previous work at Moorea, French Polynesia, uncovered five co-occurring broadcast spawning Pocilloporid species in the fore reef habitat alone that can only be identified reliably using verified genetic markers (Johnston, Cunning, et al., 2022).These cryptic species differed in their relative abundance on the fore reef slope across depths of only 5, 10, and 20 m (Johnston, Wyatt, et al., 2022).Differences in relative abundance across depths on the relatively steep fore reef slope (all accessible in a single dive) were greater than differences among sites separated by several kilometers.
Therefore, we hypothesized that the well-known environmental differences among structural habitat zones typical within coral

| Sites and sampling design
We sampled Pocillopora colonies from five different habitats at each of four sites (sites 1, 2, 4, and 5) of Moorea (Figure 1).Site locations and names correspond to those used by the Moorea Coral Reef Long-Term Ecological Research (MCR-LTER) program (Holbrook et al., 2018).At each site, the five habitats were: (1) fringing reef, (2) back reef lagoon, (3) fore reef at 5 m depth, (4) fore reef at 10 m depth, and ( 5) fore reef at 20 m depth.The fringing reef sites were ~1 m deep and ~20-30 m from shore at sites 1 and 2, 60 m from the shore at site 4, and 140 m from the shore at site 5.The back reef lagoon sites were ~1-2 m deep and typically ~600-900 m from the shore.The fore reef sites have slope angles ranging from 33% to 55% among sites (Leichter et al., 2012) and were typically 1000-1200 m from shore.
At each habitat at each site (except the fore reef 10 m), 50 cm × 50 cm quadrats (as many as was logistically possible) were randomly placed on the reef along the target depth contour to estimate relative abundance.The samples from the fore reef 10 m habitat came from colonies whose tissue was sampled in 2019 (24 70 cm × 70 cm quadrats per site) from Burgess et al. (2021) and were confirmed to still be alive in censuses conducted in December 2021 (hence smaller sample sizes).Except for the fore reef 10 m, samples from all habitats at sites 1 and 2, and from the fore reef at sites 4 and 5 were collected in December 2021.Samples from the fringing reef and back reef lagoon at sites 4 and 5 were collected in December 2022.There was no obvious disturbance (e.g., COTS, cyclone) or environmental event (e.g., marine heatwave) between December 2021 and December 2022.Only samples collected in December 2021 were used to assess symbiont identities, to avoid the potential influence of different sampling periods on symbiont assemblages.Tissue (~1 cm) from every Pocillopora colony within the quadrat was sampled (Table 1), which meant we did not target specific morphologies that could bias results.Tissue was collected from a total of 724 Pocillopora colonies.Tissue was collected using small bone clippers and stored in DMSO buffer or DNA/RNA Shield (Zymo Research).

| DNA extraction and quantification
Genomic DNA was extracted from tissue using the OMEGA (Bio-Tek) E.Z.N.A. Tissue DNA Kit or Chelex 100 Resin (Bio-Rad).OMEGA DNA extractions were used for all samples from December 2021 and processed for the genetic identification of Symbiodiniaceae.For the OMEGA extractions, three elutions (50, 100, and 100 μL) were collected in HPLC grade H2O.After inspection on a 1% agarose gel, all three elutions were combined.Extractions were quantified using the Qubit dsDNA HS Assay kit with the Qubit Fluorometer (ThermoFisher Scientific).For the Chelex extractions, 10 μL of DNA Shield from which the sample was preserved was placed into 100 μL of 10% Chelex solution and incubated at 55°C for 60 min followed by 95°C for 20 min.
Pocillopora meandrina and P. grandis both share mtORF haplotype 1, but are distinct species (Johnston et al., 2017;Johnston, Cunning, et al., 2022), so to distinguish these two species we used a restriction fragment length polymorphism (RFLP) gel-based assay following Johnston et al. (2018).DNA from samples of mtORF haplotype 1 was used for PCR amplification of the histone 3 region using the PocHistone primers PocHistoneF and PocHistoneR detailed in Johnston et al. (2018).PCR mixes were prepared as described above using the following thermocycling protocol: an initial denaturation step of 60 s at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 53°C, and 60 s at 72°C, and a final 5-min extension step at 72°C.Five microliters of PCR product was then digested with 0.5 μL of XhoI restriction enzyme (New England BioLabs, Ipswich, MA, USA), 0.8 μL of 10× CutSmart buffer, and 2.7 μL of deionized water, for a final volume of 9 μL for 1 h at 37°C, followed by heat inactivation for 20 min at 65°C (Johnston et al., 2018).Three microliters of the digested products were run on a 1% or 2% TAE-agarose gel at 70 V.
Samples that are P. meandrina present as a single band on the gel around ~700 bp, while samples that are P. grandis present as two bands around ~400 and 300 bp.Sometimes P. grandis presents as three bands (at ~700, ~400, and ~300 bp), and our sequencing of the histone 3 region consistently confirms that these samples are P. grandis, and that three bands likely arises from incomplete cutting of the restriction enzyme.
Importantly, we have previously validated the use of the mtORF and histone 3 markers as suitable species-level markers to delineate Pocillopora species at Moorea (Johnston, Cunning, et al., 2022).In that study, we used multiple genomic datasets (complete mitochondrial genomes, nuclear genomic loci, and thousands of linked and unlinked genome-wide single nucleotide polymorphisms) and phylogenetic approaches (Bayesian phylogenetic trees, SNAPP species trees, and discriminant analysis of principal components), in combination with algal symbiont identity and distribution patterns, to identify distinct evolutionary lineages of Pocillopora that are considered to be taxa on the basis of genetically distinct groups occurring in sympatry (De Queiroz, 2007).At Moorea, mtORF haplotype 1 P. meandrina and haplotype 8, despite being distinct mitochondrial lineages (differing by seven base pairs), are not differentiated when using (nuclear) genomewide single nucleotide polymorphisms (SNPs) (Johnston, Cunning, et al., 2022).Therefore, colonies identified as mtORF haplotype 8 were included with colonies identified as P. meandrina, which was also justified based on their similar within-reef distribution patterns and responses to heatwaves (Burgess et al., 2021;Johnston, Cunning, et al., 2022).Our organization of mtORF haplotypes into taxa based on genome-wide data is consistent with subsequent studies (Oury et al., 2023;Voolstra et al., 2023) that have also confirmed the suitability of the mtORF marker (plus histone marker in the case of mtORF haplotype 1) using complementary species delimitation approaches on genome-wide SNPs (Table 2).

TA B L E 1
The number of samples analyzed using mtORF (for Pocillopora species identification), ITS2 (for Symbiodiniaceae identification), and psbA ncr (for Cladocopium species identification) in each habitat and site.

| Genetic identification of Symbiodiniaceae
Extracted DNA from 377 samples was used for PCR amplification of the ribosomal internal transcribed spacer 2 (ITS2) region using the SYM_VAR primer pairs (Hume et al., 2018).Initial PCR was performed using Phusion High-Fidelity MasterMix (ThermoFisher Scientific) with an initial denaturation step at 98°C for 2 min, followed by 35 cycles of 98°C for 10 s, 56°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for 5 min.Amplified DNA was cleaned using AMPure XP beads and used as template for an index PCR in which unique combinations of Nextera XT index primers were used for each sample.Eight cycles of PCR were performed as described above with an annealing temperature of 55°C.Libraries were then cleaned, normalized, and pooled for sequencing on an Illumina MiSeq platform with 2× 300 paired-end reads at Florida State University.We included four technical replicates (i.e., four samples where the PCR was performed twice), one on each 96-well plate.
Demultiplexed forward and reverse fastq files were passed remotely to SymPortal (Hume et al., 2018), which removed non-Symbiodiniaceae sequences and then grouped Symbiodiniaceae sequences by genera.Being a ribosomal gene, there are usually multiple ITS2 copies within a single Symbiodiniaceae cell.ITS2 sequence variation can, therefore, come from intragenomic variation among gene copies, in addition to intergenomic variation among Symbiodiniaceae genotypes.To parse ITS2 sequence variation into intra-and intergenomic variation, SymPortal identifies repeatedly co-occurring sequences with similar relative abundances as "defining intragenomic variants" (DIVs), which are then collapsed into ITS2-type profiles that may represent distinct taxonomic units above, at, or below the species level (Hume et al., 2018).The full (post-quality filtering) diversity of ITS2 sequence variation was used for downstream statistical analysis, while type profiles were used to aid visualization only.
Since ITS2 is not a species-level marker, we also used the noncoding plastid minicircle (psbA ncr ) to distinguish between Cladocopium species (Moore et al., 2003) for 326 Pocillopora colonies (Table 1).The  & LaJeunesse, 2011).A Bayesian phylogeny of this larger data set was generated using beast version 2.6.2 (Bouckaert et al., 2019).We used the GTR model of evolution, a random local clock, and the birth-death model as the tree prior.The MCMC was run for 100,000,000 genera-

Note:
The association of mtORF haplotypes and species names with species hypotheses identified in other studies using genome-wide data is also provided for comparison and consistency across studies.
a Taxonomic description in Johnston and Burgess (2023).
TA B L E 2 Summary of the mtORF haplotypes that were used to identify Pocillopora species, and their associated names, following Johnston, Cunning, et al. (2022).
was not the focal species.Then, we fit binomial generalized linear models (glm) using glmmTMB v1.1.9(Brooks et al., 2017) with a logit link function and habitat as a fixed effect to test if the proportion of colonies belonging to a given species in each habitat (i.e., relative abundance of that species) varied across habitats.Model residual diagnostics were assessed using DHARMa v0.4.6 (Hartig, 2022).
To test for the effect of habitat, we used log-likelihood ratio tests where the deviance was assessed against a χ 2 distribution.Because not every species was found in every combination of habitat and site, we could not statistically assess the interactive or additive effects of site on the patterns of relative abundance across habitats within each species.As a result, sites were pooled for the binomial glm, but the raw proportions presented in Figure S1 show similar relative abundance patterns across sites.
To analyze variation in the composition of Pocillopora species among habitats, we used distance-based redundancy analysis (dbRDA) implemented in the vegan v2.6-6.1 package in R (Oksanen et al., 2022).The principal coordinates were estimated using Bray-Curtis dissimilarity matrices (based on the proportion of individuals belonging to each species in each site and habitat).Habitat was the constraining variable.The effects of habitat were determined using an ANOVA-like permutation test with 99,999 permutations implemented in anova.cca.Ordination biplots were used to visualize results and aid in interpretation.
To visualize differences in Symbiodiniaceae ITS2 sequence diversity between Pocillopora species, we used principal coordinate analyses (PCoA) implemented using cmdscale function in the R package vegan with a Bray-Curtis dissimilarity measure.Differences among species were tested using permutational multivariate analysis of variance (PERMANOVA) with 99,999 permutations, implemented using the adonis2 function in vegan.
We also tested for differences in ITS2 sequence diversity among habitats using PCoA and PERMANOVA for C. pacificum (based on psbA ncr sequences and ITS2 type profiles dominated by C1d and C1ag) when hosted by P. tuahiniensis colonies, and for C. latusorum (based on psbA ncr sequences and ITS2-type profiles dominated by C42a and C42g) when hosted by P. meandrina colonies.Only habitats with 10 or more samples were included, which also meant that there were not enough samples for this analysis in other Pocillopora species.
To estimate niche breadth, we used the measure proposed by Levins (1968), which estimates the uniformity of the distribution of individuals among resource states (Carscadden et al., 2020).We estimated reef habitat niche breadth by considering resource states to be the five reef habitats, and symbiont niche breadth as the 475 ITS2 sequence types recovered in the post_med output from SymPortal.

Niche breadth was then estimated as
And standardized to a scale from 0 to 1 by To estimate niche overlap among cryptic Pocillopora species, we used the Horn-Morisita index (Horn, 1966) where p ij is the proportion of reef habitat, or ITS2 sequence type, i used by species j. p ik is the proportion of reef habitat, or ITS2 sequence type, i used by species k.R is the total number of reef habitats or ITS2 sequence types.The Horn-Morisita index is interpreted as the probability that two individuals drawn randomly from each of two species will both belong to the same species, relative to the probability of randomly drawing two individuals of the same species from each species alone (Horn, 1966).The index varies from 0, when the two species are associated with completely different reef habitats or ITS2 sequence types, to 1 when two species are equally associated with exactly the same reef habitats or ITS2 sequence types.
Note that the Horn-Morisita measure of overlap measures the extent to which the niche space of species k overlaps that of species j, so is not symmetrical between species.We chose to present niche breadth and overlap estimates as boxplots to show variation among sites and individuals.(Figure 2; Table 3).The habitat that P. tuahiniensis, P. meandrina, and P. verrucosa were most common differed among each of these species.The patterns of relative abundance across habitats when all sites were pooled generally reflected the patterns within each site (Figure S1).The notable exception was the fringing reef habitat at site 4, in which P. meandrina, P. verrucosa, and P. grandis were

TA B L E 3
Statistical results for the effect of habitat on the proportion of Pocillopora colonies belonging to each genetically identified species in each habitat from binomial generalized linear models.
The difference in relative abundance patterns among species led to the composition of Pocillopora spp.varying among habitats (F 4,15 = 10.56,p < 0.0001; Figure 3).The Pocillopora community was most unique at either end of the habitat gradient: the fringing reef and 20 m on the fore reef (Figure 3).The Pocillopora community in the fringing reef was characterized by relatively high abundance of P. acuta, the exception being the fringing reef at site 4 (see also  Cladocopium clades tended to host multiple ITS2-type profiles, with varying degrees of overlap (Figure S4).ITS2 sequence C1d is usually diagnostic of C. pacificum (Turnham et al., 2021), but our data show that ITS2 sequence C1ag can also be considered diagnostic of C. pacificum based on comparisons to samples that also had psbA ncr sequences (Figure 5; Figure S4).Specifically, and all in P. verrucosa,  S4).
Based on a combination of psbA ncr sequences and ITS2-type profiles, 96.79% of P. meandrina colonies hosted C. latusorum, while the remaining 3.21% hosted Cladocopium C116 spp.(which were all from the 5 m fore reef habitat) (Figure 5; Table 4).Samples with C116 ITS2-type profiles produced poor quality psbA ncr sequences and could not be aligned with those from C. latusorum or C. pacificum.
Pocillopora colonies with Cladocopium C116 spp.were also found in P. grandis (n = 1 from 5 m fore reef), P. tuahiniensis (one from 5 m and one from 20 m fore reef), and P. acuta (n = 1, which was the only P. acuta colony sampled from the back reef).

F I G U R E 3
Composition patterns of Pocillopora species in each habitat (colors) and site (numbers).Ordination biplot shows the first two axes of a distance-based redundancy analysis (dbRDA) using the habitat type as the constraining variable.Species scores are plotted in grey, indicating the relative contribution of each species in causing differences in the community composition among the 20 sampling locations.
-1.5 -0.5 0.     both psbA ncr and ITS2-type profiles in four colonies (Figure 5), and by ITS2-type profiles in the other four colonies.One P. tuahiniensis Based on ITS2 sequences, the majority of P. acuta colonies (95%) hosted D. glynnii (Table 4), five of which also hosted C. pacificum and one also hosted C. latusorum (based on psbA ncr ).

| Genetic variation in symbiont species within each host species across habitats
For C. pacificum hosted in P. tuahiniensis, ITS2 sequence diversity overlapped among colonies sampled from the back reef versus 20 m on the fore reef (Figure 6a).However, ITS2 sequence diversity for C. latusorum differed among P. meandrina colonies sampled from the back reef and 5 m on the fore reef versus 20 m on the fore reef (Figure 6b).
Durusdinium glynnii was hosted by P. acuta, and to a lesser extent P. verrucosa (Table 4) that were sampled from the fringing

| Niche breadth and overlap
Pocillopora meandrina, which cannot reliably be differentiated from P. tuahiniensis or P. verrucosa in the field, had the broadest reef habitat niche breadth (Figure 7), because it was relatively more common in all habitats (Figure 2).Pocillopora meandrina had a high niche overlap with its sister species P. grandis (Figure 8).
Pocillopora verrucosa had a similar niche breadth to sister species P.
tuahiniensis, but the former was most common in shallow habitats (1 m fringing, 2 m back reef) and the latter most common in deep habitats (20 m on the fore reef) (Figure 8).Pocillopora acuta had the narrowest niche breadth because it was largely restricted to the fringing reef (Figure 2).
All Pocillopora species generally had narrow niche breadths in terms of the symbiont identities they associate with (based on ITS2 sequence diversity).However, the rank order of symbiont niche breadths among Pocillopora species was similar to that for habitat niche breadths.As a result, there was a positive correlation between reef habitat niche breadth and symbiont niche breadth across Pocillopora species.That is, Pocillopora species associating with a broader range of ITS2 DIV's also occupied a broader range of reef habitats.

| DISCUSS ION
We present an extensive dataset on the fine-scale niche differences among morphologically cryptic species, the results of which have important implications for the conservation and restoration of coral populations.By using genetic markers to properly identify species of both the coral host (724 colonies) and the symbiont (from 423 colonies [58%]), we uncovered niche differences among the common group of Pocillopora corals that cannot be reliably identified to species in the field based on morphology and have typically been lumped together or misidentified in previous studies.We found that the relative abundance of Pocillopora species differed across habitats within the reef-that is, while most species can be found in most habitats, no two Pocillopora species had the same distribution-abundance pattern across the same habitats.In general, cryptic Pocillopora species have shifted their realized niche space relative to each other while maintaining similar dispersion of niche positions among their individuals.Sister taxa P. verrucosa and P. tuahiniensis (Johnston & Burgess, 2023;Johnston, Cunning, et al., 2022) had similar niche breadths, and hosted the same specialist symbiont species (mostly Cladocopium pacificum), but the former was most common in shallow habitats and the latter most common in deep habitats.
In contrast, sister taxa P. meandrina and P. grandis had overlapping niches and high niche breadths, and hosted the same specialist symbiont species (mostly C. latusorum), though P. meandrina was much more common overall than P. grandis.Pocillopora cf.effusa tended to host the same symbiont species as P. meandrina and P. grandis, but exhibited a narrower niche breadth, being relatively more common on the fore reef than the back reef (and absent on fringing reefs).Reef habitat niche overlap (Horn-Morisita index) species with which P. acuta overlapped the most were relatively rare (P.grandis and P. verrucosa).We also uncovered a relationship between reef habitat niche breadth and symbiont niche breadth across Pocillopora species, such that Pocillopora species associating with a broader diversity of symbionts (ITS2 sequence diversity) also occupied a broader diversity of reef habitats.Overall, our results support the hypothesis that structural habitat zones within coral reefs, not just depth, play an important role in the generation and coexistence of cryptic Pocillopora species and their associated Symbiodiniaceae.
The patterns of relative abundance we uncovered could be driven by several mechanisms that are not mutually exclusive (Grosberg & Levitan, 1992;Hughes et al., 2000).One hypothesis is that larvae are capable of dispersing among all habitats, and all species recruit at equal densities in all habitats, but post-settlement survival favors different species in different habitats where species traits, including symbiont identity and physiology, best match the local environmental conditions (Edmunds et al., 2024;Prada & Hellberg, 2014;Rippe et al., 2021), such as light and temperature variability (Johnston, Wyatt, et al., 2022).Our results suggest that symbiont identity might not be that important for explaining the relative abundance of hosts (except perhaps in P. acuta), since the same symbiont species occurred in hosts with different relative abundance patterns.
However, the genetic diversity in symbionts may be an important driver or outcome of the relative abundance patterns of Pocillopora hosts.Furthermore, the local environment does not just relate to the physical environment, such that species may have the same fundamental niche, but competition, corallivory, or pathogens in each habitat causes differences in their realized niche after larval dispersal (Howe-Kerr et al., 2023;Huertas et al., 2021).A second hypothesis is that post-settlement survival does not differ among species or habitats, but rather a species is relatively more abundant where it has relatively higher larval settlement (Gaines & Bertness, 1992).Species differences in larval settlement among habitats could arise through larval transport, which could occur if species spawn at different times with different current speed and direction, or through larval behavior and habitat selection (Edelaar et al., 2008;Mulla et al., 2021;Schmidt-Roach et al., 2012).Our preliminary data on recruitment onto settlement tiles suggest that at least P. acuta, with brooded larvae and larval dispersal over the scales of meters (Gorospe & Karl, 2013), does not recruit at fore reef locations (Burgess unpublished data).A third hypothesis is that the recruitment and environmental suitability among habitats do not differ among species, but the current distribution reflects the history of disturbance and recovery cycles.We consider this third hypothesis least likely for most species because coral cover was reduced to <1% by 2010 at Moorea and increased steadily from sexual recruitment until 2019 (Edmunds, 2018).However, the marine heatwave in 2019 caused much higher mortality on the fore reef and in P. cf. effusa compared to that in other Pocillopora species (Burgess et al., 2021).Prior to 2019, it was likely that P. cf. effusa was at least equally dominant with P. meandrina and P. tuahiniensis on the fore reef (Burgess et al., 2021).Therefore, an important question now is understanding the relative contribution of multiple processes facilitating niche partitioning (e.g., Johnston, Wyatt, et al., 2022).
Although C. latusorum and C. pacificum are mostly host specialists, similar to that reported previously (Johnston, Cunning, et al., 2022;Turnham et al., 2021), our results show that, by sampling hundreds of colonies and using multiple genetic markers, there is more flexibility than has previously been documented.(Guadayol et al., 2014;Johnston, Wyatt, et al., 2022;Reid et al., 2019;Wyatt et al., 2020Wyatt et al., , 2023)), and ignoring such fine-scale environmental variation among habitats or depths within reefs is likely to provide an incomplete understanding of how the environment affects the distribution of genetic and species diversity, and symbioses, that are relevant to understanding acclimatization and adaptation.Furthermore, sampling Pocillopora species based on in situ morphological assessment will provide an incomplete picture at the species level, not only because such sampling will likely reveal multiple genetic species within a morphological group (Oury et al., 2023, Voolstra et al., 2023), but it will miss individuals that are the same genetic species but not sampled because they were morphologically different.What might be considered a large, widespread species based on morphological indicators, may instead be many species with populations that are smaller, geographically restricted, specialized to different habitats within reefs, and more prone to extinction than previously thought (Meziere et al., 2024).
The presence of cryptic species specialized to different habitats Especially because most restoration programs target a particular morphological group (Hughes et al., 2023), unknowingly collecting or growing fragments from a species with the highest capacity for survival and reproduction in fore reef habitats and attempting to use those fragments to restore populations in back reef habitats, for example, will be less successful than using a species most suited to back reef habitats.Similarly, using fragments from a cryptic species that is particularly susceptible to bleaching (e.g., P. cf. effusa; Burgess et al., 2021) would lead to decreased success rates, especially if heatwaves were responsible for depleting coral populations in the first place.Restoration practices that translocate corals or their offspring among habitats and ignore cryptic species are also prone to unintentionally reconfiguring species-environment specializations and manipulating gene flow that could negatively affect near-future adaptive capacity, and ultimately do more harm than good.On the other hand, restoration programs that actively target cryptic species and deliberately incorporate diverse habitats are likely to have higher success because a given morphological group will contain the available portfolio of genotypes, rather than a narrow subset, which will be especially important when the particular species or genetic variants that will survive and reproduce in the near future is unpredictable (Colton et al., 2022;Webster et al., 2017).It is, therefore, critical that restoration practitioners use sound knowledge and principles to maximize positive outcomes, even if restoration is at best only likely to be successful at very small spatial and temporal scales (Hughes et al., 2023).
Our results also have important implications for understanding how temporary micro-refuges within reefs could underlie recovery at reef scales.Disturbances that threaten coral reefs, such as heatwaves, cyclones, Crown-of-Thorns seastar outbreaks, pollution, and over-fishing occur over regional scales but are also spatially variable at small scales within reefs (Baird et al., 2018;Crosbie et al., 2019;Penin et al., 2007;Sully & van Woesik, 2020;Yadav et al., 2023).Especially in relation to coral bleaching, small-scale environmental "refuges" associated with depth (Baird et al., 2018;Crosbie et al., 2019;Penin et al., 2007), distance from shore (Matias et al., 2023;Sully & van Woesik, 2020), or back reef pools (Bay & Palumbi, 2014;Thomas et al., 2018) are common.For example, the marine heat wave at Moorea (French Polynesia) in 2016 affected corals on the back reef much more than the fore reef (Donovan et al., 2020), whereas the heat wave in 2019 affected corals on the fore reef much more than the back reef (Burgess et al., 2021).Such differences are likely mediated by higher nutrients in the back reef compared to the fore reef (Leichter et al., 2013), and the dynamics of depth-dependent internal wave cooling on the fore reef (Wyatt et al., 2020(Wyatt et al., , 2023)).The implications of our results are that habitatspecific disturbances or environmental changes will affect some cryptic species more than others, which could lead to resilience via response diversity (Baskett et al., 2014;Burgess et al., 2021), or simply elevate the extinction risk in vulnerable species in certain habitats.Such habitat-abundance associations would also lead to species responses to habitat-specific disturbances that are potentially independent of any intrinsic differences in the physiological responses among different species.Furthermore, it will be difficult to discern whether differences in the response to the environment or disturbance among habitats at the genus level (i.e., without genetically identifying colonies to species and lumping them together) are driven by environmental conditions of the habitat or the cryptic species that dominates there.
Our ability to anticipate the consequences of climate change and restoration interventions will depend on understanding how genetic diversity is partitioned among species that are morphologically indistinguishable but ecologically and evolutionarily distinct (Colton et al., 2022;McManus et al., 2021;Webster et al., 2017).In that light, the identification and habitat associations of cryptic species and their algal symbionts here is important because we have uncovered niche differences within one of the most common group of corals (Pocillopora) that cannot be reliably identified to species in the field based on morphology and have typically been lumped together or misidentified in the past.An important implication is that researchers using transplant experiments between depths or habitats could misinterpret their results as being caused by depth or habitat, when they are instead due to differences among cryptic species.Similarly, assessment of treatment effects on a purported species of Pocillopora will be misleading if analyses are not based on the identification of colonies to species using genetics.Previous results on the ecology of Pocillopora at the species level should be considered unreliable if samples were not genetically identified to species, and future studies should endeavor to genetically identify samples to species when studying the population and community ecology of Pocillopora.
reefs are strong enough to generate niche differences among cryptic Pocillopora species, and this would be reflected by different relative abundances of host species among habitats and the identity and diversity of the photosynthetic algal symbionts they host.We sampled Pocillopora corals across five habitats (the fringing reef, back reef, 5 m on the fore reef, 10 m on the fore reef, and 20 m on the fore reef slope) extending seaward from the shore at each of four sites separated by several kilometers around the island of Moorea, French Polynesia.Specifically, we (1) quantified the relative abundance of genetically identified Pocillopora species across habitats, (2) tested for associations between coral host (Pocillopora) and symbiont (Symbiodiniaceae) identity, (3) tested how habitat type affected genetic variation in symbionts, and (4) quantified niche breadth and divergence among Pocillopora species and the extent to which habitat niche breadth correlated with symbiont niche breadth among Pocillopora species.
psbA ncr region was amplified using the 7.4-Forw and 7.8-Rev primers and protocol ofMoore et al. (2003), and the region was sequenced in both the forward and reverse directions.Sequences were aligned and manually checked in Geneious Prime.To place the clades in our current study, we included five sequences from Johnston, Cunning, et al. (2022), one from each of the Cladocopium clades found in that study, and as outgroups included two sequences of C. goreaui from Turnham et al. (2021) and three sequences of Cladocopium spp.collected from Pocillopora cf.effusa colonies from Clipperton Atoll (Pinzón where p j is the proportion of individual colonies from a given Pocillopora species found in habitat j, or the proportion of ITS2 sequence types j in each individual colony for a given Pocillopora species.A niche breadth of 0 occurs when a species is only found in one habitat, or an individual colony is only associated with one ITS2 sequence type.A niche breadth of 1 occurs when the relative abundance of a species is equal across all habitats, or individual colonies host an equal amount of all ITS2 sequence types. ik relatively more common than P. acuta, which dominated the fringing reef habitat of the other three sites.Pocillopora tuahiniensis was relatively more common at 20 m on the fore reef, and its abundance relative to other Pocillopora species decreased sharply from only 20 to 10 m.Pocillopora verrucosa, to which P. tuahiniensis is most closely related phylogenetically and which are morphologically similar, exhibited the opposite pattern, being relatively more common in the back reef lagoon and fringing habitat, while relatively rare on the fore reef.Pocillopora meandrina, which cannot reliably be differentiated from P. tuahiniensis or P. verrucosa in the field, had a "hump-shaped" relative abundance pattern extending from the shore, peaking at the 5 m fore reef habitat, unlike the other species.Pocillopora grandis exhibited no change in relative abundance across habitats, except at 20 m depth on the fore reef where its relative abundance was lowest.Pocillopora cf.effusa, which most closely resembles P. grandis morphologically, was relatively more common on the fore reef at all depths compared to the back reef habitat, and was not present in samples from the fringing reef.F I G U R E 2 The proportion of all Pocillopora spp.colonies belonging to each genetically identified species (rows) in each habitat (x-axis) over four sites around the island of Moorea.Images show examples of gross colony morphologies in the field (but do not necessarily show the morphological variation within species).Haplotypes refer to mtORF haplotypes.Sample sizes are shown in

Figure
Figure S1).The Pocillopora community at 20 m was characterized by relatively high abundance of P. tuahiniensis.The composition of the Pocillopora community was relatively similar across the back reef lagoon, and at 5 m and 10 m on the fore reef, because species whose relative abundance differed the most between back reef lagoon and shallow fore reef habitats (i.e., P. verrucosa, P. cf. effusa) were relatively rare overall.

3. 2 |
Figures 4 and 5).Using psbA ncr , nine Cladocopium clades were recovered, four within C. latusorum and five within C. pacificum (Figures S2and S3).Clades I through VI were well supported and recovered the clades reported inJohnston, Cunning, et al. (2022) andJohnston, Wyatt, et al. (2022), though clades VII, VIII, and IX were relatively less well supported (all within C. pacificum; see FigureS3).psbA ncr Composition patterns of Symbiodiniaceae based on ITS2 sequence diversity (i.e., the full post-quality-filtering diversity of ITS2 sequence variation using the post_med output from SymPortal).Both plots show the same ordination biplot using the first two axes of a principal coordinates analysis (PCoA).Each dot represents a single Pocillopora colony.Each colony is identified by its host species identification in (a), and by its dominant symbiont species in (b) based on a combination of psbA ncr sequences and ITS2-type profiles.Ellipses denote the standard deviation of data around the centroid.

F
Analysis of samples with both ITS2 sequences (SymPortal post-med ITS2 sequence data) and psbA ncr sequences.The top tree shows the UPGMA tree of the between-sample Generalized UniFrac distances from SymPortal.Each vertical bar represents a single coral sample, showing the Pocillopora identity derived from the mtORF and RFLP molecular assays (first row), the habitat from which the sample was collected (second row), the hosted clade of Cladocopium spp.identified from the psbA ncr sequences (FiguresS2 and S3) (third row), and ITS2-type profiles from SymPortal, colored according to type profile with grey showing the proportion of non-profile sequences found in each sample (fourth row).TA B L E 4Summary of the associations between host Pocillopora species and symbiont Symbiodiniaceae species.
colony hosting C. pacificum (based on psbA ncr ) also hosted D. glynnii (based on ITS2-type profile).Similarly, 4.26% (n = 2) of P. verrucosa colonies hosted C. latusorum, indicated by both psbA ncr and ITS2type profiles in one colony, and ITS2-type profiles in the other.Three P. verrucosa colonies hosted D. glynnii, but did not produce psbA ncr sequences.

E 6
Principal coordinate analyses (PCoA) of Symbiodiniaceae ITS2 sequence diversity among habitats (denoted in legend) for (a) Cladocopium pacificum (mostly psbA ncr clades IX and VI) hosted by Pocillopora tuahiniensis (n = 21 at 2 m, n = 59 at 20 m), and (b) C. latusorum (mostly psbA ncr clade I) hosted by P. meandrina (n = 44 at 2 m, n = 85 at 5 m, n = 11 at 20 m).Sample size (n) and PERMAMOVA results (F = Fvalue, df = degrees of freedom, p = p-value) are presented at the top of each plot.Ellipses denote the standard deviation of data around the centroid.Note that two outlier samples were removed from each analysis to ensure they were not driving the differences among habitats.Levin's niche breadth estimates for each Pocillopora species in terms of (a) reef habitats and (b) symbionts (ITS2 sequence diversity, DIV).Boxplots show the spread of niche breadth estimates across each of the four sampling sites in (a), and among each individual coral colony in (b) (sample sizes in Table1).(c) shows the correlation between the species mean reef habitat niche breadth and the species mean symbiont niche breadth.Error bars denote standard error.ρ = Spearman's rank correlation coefficient, p = p-value.
the 20 m fore reef habitat, D. glynnii was only found in two colonies (P.tuahiniensis and P. cf. effusa).Cladocopium C116 spp. was only found in colonies from the back reef, 5 m fore reef, and 20 m fore reef.Symbiodinium microadriaticum was only found in P. grandis from shallow habitats: the fringing reef, back reef, and 5 m fore reef.
within a reef system has particularly important implications for coral restoration.The most common method of coral restoration involves transplanting fragments of coral colonies to the reef, either directly from existing fragments or from coral gardening (where small fragments are raised in nurseries prior to outplanting)(Boström- Einarsson et al., 2020).Corals in the genus Pocillopora are one of the most common corals used in restoration(Boström-Einarsson et al., 2020;Hughes et al., 2023), but are often identified as species based on morphology, or are all considered as a single species.
Site numbers correspond to those used for the MCR LTER.Numbers are presented in the following format: mtORF/ITS2/psbA ncr .

Species name mtORF haplotype Oury et al. (2023) Genomic species hypotheses (GSH) Voolstra et al. (2023) SVDquartet lineage
To quantify how the relative abundance of a given species differed across environments, we first assigned a colony a value of 1 if it was the focal species, or 0 if it

3 | RE SULTS 3.1 | Relative abundance of cryptic host species (Pocillopora) across habitats
From extensive sampling of 724 Pocillopora colonies from around the island of Moorea, there were six sympatric species of Pocillopora: P.
Table 1 and statistical results are shown in Table3.Note the different y-axis scales between each species.The proportion and 95% confidence interval were estimated from a binomial generalized linear model.
Note: Statistical tests for Pocillopora acuta are not included because all but one colony was sampled from the fringing reef.
Schmidt-Roach et al., 2012)ly brooding species (the other species are broadcast spawners;Schmidt-Roach et al., 2012)and had the narrowest niche breadth, being restricted to the fringing reef habitat, where unlike other host species, most colonies hosted the generalist, Niche overlap between a given species (a-f) with other species, in terms of its reef habitat niche distribution.Niche overlap is measured using The Horn-Morisita index.Each plot shows the extent to which the relative abundance distribution of that species overlaps with the other species.
and thermally tolerant, symbiont species Durusdinium glynnii.Other F I G U R E 8